1260 infinity uv detector

HPLC–MS was performed on an Agilent 1260 system with 1260 Infinity UV detector and a Bruker microQ-TOF II mass spectrometer equipped with an ESI ionization probe. The innoval C18 column (4.6 × 250 mm; 5 μm particle size) was used in this study. The HPLC conditions for isovaleryl-CoA, HIBP and PIVP were as follows: solvent A = H₂O (containing 20 mM ammonium acetate for the analysis of isovaleryl-CoA, or containing 0.1 % formic acid for the analysis of HIBP and PIVP); solvent B = methanol; flow rate = 1 mL/min; 0–5 min 95 % A and 5 % B, 6–45 min 95 % A and 5 % B to 100 % B (linear gradient). All these products were detected at 254 nm. Standard calibration curve of isovaleryl-CoA was generated with a series of known concentrations of the isovaleryl-CoA standard purchased from Sigma-Aldrich (Milwaukee, WI, USA). Standard calibration curves of HIBP and PIVP were generated with a series of known concentrations of the purified compounds from large scale fermentation. All experiments were carried out in triplicate and repeated at least twice. The titer was presented as mean ± SD.